Many people with diabetes have normal levels of insulin in their blood, but the insulin fails to stimulate muscle cells and fat cells in the normal way (type II diabetes). Currently it is believed that there is a breakdown in the mechanism through which insulin signals to the muscle and fat cells.
Protein phosphorylation and dephosphorylation are fundamental processes for the regulation of cellular functions. Protein phosphorylation is prominently involved in signal transduction, where extracellular signals are propagated and amplified by a cascade of protein phosphorylation and dephosphorylation. Two of the best characterized signal transduction, where extracellular signals are propagated and amplified by a cascade of protein phosphorylation and dephosphorylation. Two of the best characterized signal transduction pathways involve the c-AMP-dependant protein kinase (PKA) and protein kinase C(PKC). Each pathway uses a different second messenger molecule to activate the protein kinase, which, in turn, phosphorylates specific target molecules.
A novel subfamily of serine (Ser)/threonine (Thr) kinases has been recently identified and cloned, termed herein the RAC-PK [see Jones et al., Proc Natl Acad Sci USA, Vol. 88, No. 10, pp. 4171-4175 (1991); and Jones, Jakubowicz and Hemmings, Cell Regul, Vol. 2, No. 12, pp. 1001-1009 (1991)], but also known as RAC-PK or Akt. RAC kinases have been identified in two closely-related isoforms, RACα and RACβ, which share 90% homology at the gene sequence. Mouse RACα (c-akt) is the cellular homologue of the viral oncogene v-akt, generated by fusion of the Gag protein from the AKT8 retrovirus to the N-terminus of murine c-akt. Human RACβ is found to be involved in approximately 10% of ovarian carcinomas, suggesting an involvement of RAC kinases in cell growth regulation.
Another kinase implicated in cell growth control is S6 kinase, known as p70S6K. S6 kinase phosphorylates the 40S ribosomal protein S6, an event which up-regulates protein synthesis and is believed to be required in order for progression through the G1 phase of the cell cycle. The activity of p70S6K is regulated by Ser/Thr phosphorylation thereof, and it is itself a Ser/Thr kinase. The p70S6K signaling pathway is believed to consist of a series of Ser/Thr kinases, activating each other in turn and leading to a variety of effects associated with cell proliferation and growth. RAC-PK is believed to lie on the same signaling pathway as p70S6K, but upstream thereof.
RAC kinases contain an amino-terminal pleckstrin homology (PH) domain. See Haslam, Koide and Hemmings, Nature, Vol. 363, No. 6427, pp. 309-310 (1993). The PH domain was originally identified as an internal repeat, present at the amino and carboxy-termini of pleckstrin, a 47 kDa protein which is the major PKC substrate in activated platelets. See Tyers et al., Nature, Vol. 333, No. 6172, pp. 470-473 (1988). The superfamily of PH domain containing molecules consists of over 90 members including Ser/Thr kinases, e.g., RAC, Nrk, β-adrenergic receptor kinase (βARK) and PKC.mu.; tyrosine kinases, e.g., Bruton's tyrosine kinase (Btk), Tec and Itk; GTPase regulators, e.g., ras-GAP, ras-GRF, Vav, SOS and BCR; all known mammalian phospholipase Cs; cytoskeletal proteins, e.g., β-spectrin, AFAP-110 and syntrophin; “adapter” proteins, e.g., GRB-7 and 3BP2; and kinase substrates, e.g., pleckstrin and IRS-1.
While the PH domain structure has been solved for β-spectrin, dynamin and pleckstrin's amino-terminal domain, its precise function remains unclear. The presence of PH domains in many signaling and cytoskeletal proteins implicates it in mediating protein-protein and membrane interactions. Indeed, the PH domain of the βARK appears partly responsible for its binding to the β.gamma.-subunits of the heterotrimeric G-proteins associated with the β-adrenergic receptor, while the PH domain of the Btk appears to mediate an interaction with PKC. Several PH domains have been shown to be able to bind phosphatidyl-inositol-4-5-bisphosphate in vitro, although weakly.
IMPDH is a highly-conserved enzyme (41% amino acid identity between bacterial and mammalian sequences) involved in the rate-limiting step of guanine biosynthesis. In mammals there are two isoforms, 84% identical, called type I and type II which are differentially-expressed. See Natsumeda et al., J Biol Chem, Vol. 265, No. 9, pp. 5292-5295 (1990). Type I is constitutively-expressed at low levels while the type II mRNA and protein levels increase during cellular proliferation. IMPDH activity levels are also elevated during rapid proliferation in many cells. See Collart and Huberman, J Biol Chem, Vol. 263, No. 30, pp. 15769-15772 (1988).
By measuring the metabolic fluxes, the proliferative index of intact cancer cells has been shown to be linked with the preferential channeling of IMP into guanylate biosynthesis. Inhibition of cellular IMPDH activity results in an abrupt cessation of DNA synthesis and a cell-cycle block at the G.sub.1-S interface. The specific inhibition of IMPDH by tiazofurin and the subsequent decline in the GTP pool, results in the down regulation of the G-protein ras, which is involved in many signal transduction pathways leading to cellular proliferation. For review see Avruch, Zhang and Kyriakis, Trends Biochem Sci, Vol. 19, No. 7, pp. 279-283 (1994).
Interestingly, p53 has been implicated in regulating IMPDH activity levels. See Sherley, J Biol Chem, Vol. 266, No. 36, pp. 24815-24828 (1991). Here a moderate over-expression of p53 (3- to 6-fold) induces a profound growth arrest which is rescued by purine nucleotide precursors. Indeed, the p53 over-expression induces a specific block in IMP to XMP conversion, and a diminished activity level of IMPDH. The p53 block does not affect the rate of RNA synthesis, nor is the phenotype rescued by deoxynucleotides indicating that a lack of precursors for DNA synthesis is also not the cause of the block. It would seem most likely that this effect is mediated through a down-regulation of the GTP pool required by G-proteins, such as ras.
The above observations suggest that IMPDH type II is primarily involved in producing XMP which is channeled into the GTP pool which is crucial for the regulation of G-proteins involved in signal transduction, such as ras. It may be that the type I enzyme provides a basal level of XMP that is channeled into the GTP/dGTP pools required for RNA and DNA synthesis. Changes in IMPDH type II activity would alter the GTP/GDP ratio by specifically altering the GTP component which could greatly affect ras signaling pathways as ras is sensitive to small changes in the GTP/GDP ratio.
Glycogen synthase kinase-3 (GSK3) is implicated in the control of several processes important for mammalian cell physiology, including glycogen metabolism and the control of protein synthesis by insulin, as well as the modulation of activity of several transcription factors, such as AP-1 and CREB. GSK3 is inhibited in vitro by serine phosphorylation caused by MAP kinase and p70.sup.S6K, kinases which lie on distinct insulin-stimulated signaling pathways.
GSK3 is responsible for serine phosphorylation in glycogen synthase, whose dephosphorylation underlies the stimulation of glycogen synthesis by muscle. Thus, GSK3 inactivates glycogen synthase, resulting in an increase in blood sugar levels. Insulin inhibits the action of GSK3, which, in combination with the concomitant activation of phosphatases which dephosphory late glycogen synthase, leads to the activation of glycogen synthase and the lowering of blood sugar levels.
GSK3 is inhibited in response to insulin with a half-time of 2 minutes, slightly slower than the half-time for activation of RAC-PKα (1 minute). Inhibition of GSK3 by insulin results in its phosphorylation at the same serine residue (serine 21) which is targeted by RAC-PKα in vitro. Like the activation of RAC-PKα, the inhibition of GSK3 by insulin is prevented by phosphatidyl inositol (PI-3) kinase inhibitors wortmannin and LY 294002. The inhibition of GSK3 is likely to contribute to the increase in the rate of glycogen synthesis [see Cross et al., Biochem J, Vol. 303, Pt. 1, pp. 21-26 (1994)] and translation of certain mRNAs by insulin. See Welsh et al., Biochem J, Vol. 303, Pt. 1, pp. 15-20 (1994).
We have used the yeast two-hybrid system [see Fields and Song, Nature, Vol. 340, No. 6230, pp. 245-246 (1989); and Chien, Bartel, Sternglanz and Fields, Proc Natl Acad Sci USA, Vol. 88, No. 21, pp. 9578-9582 (1991)] to determine if RAC-PK could function by forming specific interactions with other proteins. We have identified RAC-PK as interacting with human inosine-5′ monophosphate dehydrogenase (IMPDH) type II, and with a novel protein termed RAC-PK Carboxy-Terminal Binding Protein (CTBP). RAC-PK stimulates IMPDH type II activity. In conjunction with the known role of IMPDH in GTP biosynthesis, our findings suggest a role for RAC-PK in the regulation of cell proliferation.
Moreover, using a peptide derived from GSK3 and GSK3 itself, we have been able to show that RAC-PK interacts with, phosphorylates and inactivates GSK3. This implicates RAC-PK in the regulation of insulin-dependent signaling pathways, which control cellular proliferation. Taken together, these results suggest a major involvement for RAC-PK in the control of insulin action.
Many growth factors trigger the activation of phosphatidylinositol (PI) 3-kinase, the enzyme which converts PI 4,5 bisphosphate (PIP2) to the putative second messenger PI 3,4,5 trisphosphate (PIP3) and RAC-PK lies downstream of PI 3-kinase. See Franke et al., Cell, Vol. 81, No. 5, pp. 727-736 (1995). RAC-PKα is converted from an inactive to an active form with a half-time of about 1 minute when cells are stimulated with PDGF [see Franke et al. (1995), supra], EGF or basic FGF [see Burgering and Coffer, Nature, Vol. 376, No. 6541, pp. 599-602 (1995)] or insulin [see Cross et al. (1995), supra; and Kohn, Kovacina and Roth, EMBO J, Vol. 14, No. 17, pp. 4288-4295 (1995)] or perpervanadate. See Andjelkovic et al., Proc Natl Acad Sci USA, Vol. 93, No. 12, pp. 5699-5704 (1996). Activation of RAC-PK by insulin or growth factors is prevented if the cells are pre-incubated with inhibitors of PI 3-kinase (wortmannin or LY 294002) or by over-expression of a dominant negative mutant of PI 3-kinase. See Burgering and Coffer (1995), supra. Mutation of the tyrosine residues in the PDGF receptor that when phosphorylated bind to PI 3-kinase also prevent the activation of RAC-PKα. See Burgering and Coffer (1995), supra; and Franke et al. (1995), supra.
When isolated from natural sources, especially convenient sources, such as tissue culture cells, RAC-PK and other signaling kinases are normally in the inactive state. In order to isolate active PKs, it is necessary to stimulate cells in order to switch on the signaling pathway to yield active kinase. Moreover, when cells expressing kinase enzymes are used in kinase activity assays, it is necessary to employ activating agents prior to conducting the assay. Thus, cells are normally stimulated with mitogens and/or activating agents, such as IL-2, platelet-derived growth factor (PDGF), insulin, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Such agents are expensive and, when it is desired to produce active kinases or to activate cells in large amounts, the use of such agents is disadvantageous.
Screening of candidate compounds for activity as inhibitors of RAC-PK, or other signaling kinases in order to identify candidate immunosuppressive or anti-proliferative agents requires a plentiful supply of PK. Using modern day technology, it is possible to produce large quantities of virtually any desired protein in recombinant DNA expression systems. In the case of kinases, such as those with which we are presently concerned, however, such systems are unsatisfactory because the proteins produced would be unphosphorylated and therefore inactive. There is therefore a requirement to identify a cost-effective way to produce phosphorylated PKs which can be employed in screening procedures.
It is known [see Jano et al., Biochemistry, Vol. 85, pp. 406-410 (1988)] that vanadate can activate p70S6K itself. The mechanism of this activation, however, is not known. We have now found that vanadate acts generally on signaling kinases, activating them and preventing deactivation by phosphatases. Moreover, we have found that okadaic acid, a different class of compound from vanadate which interacts with different proteins, may be used to similar effect.